1. Field of the Invention
The invention relates to a method and a device for the fixing and/or stabilization of a sample. Biomolecules are stabilized and/or tissues are fixed, and thus made durable. Biomolecules to be stabilized are in particular DNA, RNA and proteins.
2. Description of Related Art
Documents WO 03/040697 and DE 40 19 182 A1 disclose fixing tissue samples, dehydrating a fixed sample and embedding it in paraffin. The sample embedded in paraffin is cut into thin slices and studied under a microscope. According to document DE 40 19 182 A1, the impregnation of samples is supported with the aid of ultrasound. Document DE 198 20 466 A1 discloses disrupting a biological sample with the aid of ultrasound.
In order to be able to stabilize a biological sample, it is especially important for a stabilizing solution or stabilizing fluid to penetrate the sample, which contains the biomolecules, sufficiently quickly. In particular, when the biological sample is a tissue, it is advantageous for the tissue to be penetrated sufficiently quickly by a fixing solution or fixing fluid, for the tissue to be fixed successfully.
In the prior art, a manufacturer of a stabilizing solution specifies the boundary conditions that are to be observed during stabilization. Thus, a manufacturer of a stabilizing solution states, for example, what dimensions a sample must not exceed, for particular biomolecules, for example RNA, to be stabilized uniformly at every point of the sample. Such a statement may, for example, specify a certain thickness that a sample must not exceed, to achieve the desired stabilization. In some cases, other geometric specifications are also added. For example, a laboratory assistant must then, for example using a scalpel, adjust a sample to the desired size, before putting the sample in a stabilizing solution. Additionally, in many cases manufacturers state what ratio must be maintained between stabilizing solution and sample, in order to achieve a desired stabilization.
If tissues are to be fixed, often the manufacturer does not specify any boundary conditions that are to be observed for fixing, especially when the tissues are to be fixed for histological investigations. For histological investigations, as a rule formalin is used as fixing solution. The desired tissue is then placed in formalin and thus fixed.
Even if as a rule no requirements are stated for fixing with fixing solutions containing formaldehyde, for example formalin, nevertheless excessive size of a sample proves to be disadvantageous in the case of stabilization of tissue. Therefore even with formaldehyde-containing fixing solutions it is advisable not to exceed certain sizes or dimensions, in order to fix a tissue successfully.
Document U.S. Pat. No. 7,147,826 B2 discloses the provision of a closable, permeable basket, which is to be placed in another vessel containing a stabilizing solution. Liquid can penetrate into the permeable basket from all directions. If the basket contains a biological sample that is to be stabilized, it is necessary to ensure that the stabilizing solution can reach the sample from all directions.
Document U.S. Pat. No. 2003/0087423 A1 also discloses the provision of a permeable basket for a biological sample. However, in this case the basket is fastened to a lid of the vessel, to facilitate handling. Once again, it should be possible for a solution to reach a sample contained in the basket reliably and completely from all directions.
Document EP 1 262 758 A1 discloses a further permeable container for the preparation of tissue.
Cassettes made of plastic, with a hinged lid, are known from the prior art under the tradename “Histosette”, which are intended for holding tissue samples for processing or dehydration of the tissue. The area of the bottom or lid of a Histosette is at least 3 cm*2.5 cm. Said cassette is at least 0.5 cm high or thick. Bottom and lid are in the form of a sieve or are provided with a large number of slits, so that liquid can get into the cassette, for example during dehydration of a tissue.
If Histosettes are used for dehydration of tissue, for example so that histological investigations can then be carried out, tissue that has already been fixed, for example by placing in formalin beforehand, is first cut to the desired size to fit the Histosette. Then the tissue is put in the Histosette and the Histosette is closed. The Histosette together with the tissue it contains is then put in an automatic device, in which dehydration is carried out automatically.
The dehydration steps specified in said automatic device may include a repeat treatment of the already fixed tissue with a fixing solution. However, this does not mean that the tissue is fixed in the sense of the present invention, as the tissue had already been fixed previously. Then the Histosette, with the tissue it contains, is immersed successively in various alcohol baths with higher and higher alcohol concentration. The alcohol extracts the water from the tissue. To ensure gentle dehydration of the tissue, the alcohol concentration is increased very slowly from one bath to the next.
Then the Histosette, with the tissue inside, is immersed in an intermediate medium, for example xylene. The intermediate medium displaces the alcohol present in the tissue. In contrast to alcohol, the intermediate medium is miscible with paraffin, ready for the subsequent paraffin treatment.
Next, the Histosette, with the tissue inside, is immersed in hot and hence liquid paraffin. Paraffin then penetrates into the tissue. If the paraffin has penetrated into the tissue in the desired manner, the Histosette is taken out of the automatic device. The paraffin that has already hardened somewhat, with the piece of tissue inside, is then transferred to a small container or “mold” that is open at the top, and is covered with hot, liquid paraffin, so that after the paraffin has cooled, we get a paraffin block containing the tissue. When this paraffin has cooled, we have a dehydrated sample embedded in paraffin. For carrying out histological investigations, tissue sections of micrometer thickness are prepared. These tissue sections can be mounted on slides, deparaffined, stained and assessed under the microscope. One disadvantage of fixing with formaldehyde-containing fixing solutions is that biomolecules are sometimes crosslinked irreversibly and thus destroyed. As a result, the isolation of biomolecules for analytical investigations is made difficult or impossible.
A permeable cassette with a liftable lid for the accommodation of tissue samples is known from document U.S. Pat No. 2007/0140920 A1. A sample is placed into the cassette and the cassette is closed. The sample is dewatered, cleaned and infiltrated with wax in the cassette. Fixing or stabilizing the sample before the dewatering while it is present in the cassette cannot be inferred from U.S. Pat. No. 2007/0140920 A1.
Permeable cassettes for the accommodation of biological samples are also disclosed by documents U.S. Pat. No. 2005/0147538 A1 and WO 2005/037182 A2. Both documents disclose that a sample should first be fixed. Only after the fixing is sample material placed into a cassette and treated further in the desired manner.
Document U.S. Pat. No. 2006/0178598 A1 discloses a tool with protruding, needle-shaped parts provided with hooks. The protruding, needle-shaped parts should be inserted into a sample in order then to tear sample material captured by the protruding, needle-shaped parts from the rest of the sample. The tool does not comprise any walls in the form of a sieve, slit or grating.